The genomes of all living things have been colonized by parasitic genetic elements known as transposons. If left unchecked, these have the potential to produce a significant and accumulating load of mutations at each generation. Moreover, the activity of even a single transposon can cause sterility and germ cell loss in some model systems. This creates tremendous evolutionary pressure to evolve mechanisms to discriminate transposons from endogenous genes and to selectively silence the former. During the last grant period, we uncovered a small RNA-based immune system, comprising of Piwi proteins and piRNAs, that guards germ cell genomes against the activity of genomic parasites. This system contains both genetically encoded resistance (piRNA clusters) and an adaptive component (the ping-pong cycle) that focuses responses toward active elements. In this application, we propose to deepen our understanding of the composition of the piRNA pathway, to elucidate how it discriminates self (endogenous genes) from non-self (transposons), and to probe the mechanisms by which it heritably and selectively silences selfish DNA. Though proposed studies focus on Drosophila, the mechanisms, which we study, are conserved throughout metazoans. PUBLIC HEALTH RELEVANCE: The goal of this application is to further understand the biological impacts of small RNAs. Small RNAs are gene regulators that impact disease states ranging from cancer to neurodegeneration. They serve as tools for understanding normal and aberrant biological processes and will perhaps transform clinical practice by serving as the basis for a new generation of therapeutic agents.